Identification of low-frequency and rare genetic variants related to
type 2 diabetes mellitus by exome sequencing.
Type 2 diabetes mellitus (T2DM) has become a global epidemic contributing
significantly to morbidity and premature mortality. T2DM is the result from the interaction
of different factors, including environmental, genetic and acquired. It has shown that the
heredability of T2DM is around 70%. Thus far genetic studies can explain only a fraction
of the estimated genetic component of the disease. The rest of it could be explained in
low-frequency variants (MAF <0,05) and rare variants (MAF <0,01). However, the
implications of these variants in the disease have not been determined yet due to the
limitations of genetic studies conducted. Moreover, more than 90% of mutations and
polymorphisms associated with diseases are in the protein coding region, the exome.
Therefo...
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Identification of low-frequency and rare genetic variants related to
type 2 diabetes mellitus by exome sequencing.
Type 2 diabetes mellitus (T2DM) has become a global epidemic contributing
significantly to morbidity and premature mortality. T2DM is the result from the interaction
of different factors, including environmental, genetic and acquired. It has shown that the
heredability of T2DM is around 70%. Thus far genetic studies can explain only a fraction
of the estimated genetic component of the disease. The rest of it could be explained in
low-frequency variants (MAF <0,05) and rare variants (MAF <0,01). However, the
implications of these variants in the disease have not been determined yet due to the
limitations of genetic studies conducted. Moreover, more than 90% of mutations and
polymorphisms associated with diseases are in the protein coding region, the exome.
Therefore, an important fraction of low-frequency and rare variants could be found in the
exome. Finally, this project represents a novel study because of the approach and
population used.
Objectives: To identify genetic variants in the exome, including low-frequency and
rare variants, in relation to T2DM in a Spanish population.
Methodology: Exome sequencing (coverage: 20x) in 200 patients with T2DM and
200 Spanish healthy controls; all subjects had a body mass index between 25-34.9 kg/m2
and were 40 to 65 years old. Exome regions were captured and sequenced by nextgeneration
sequencing technology using Illumina HiScanSQ system to generate
2x100 bp paired end reads. A bioinformatic analysis pipeline was used to perform quality
controls, to align the reads to a reference genome and identify genetic variants. We
selected variants with MAF <20%, present in controls or cases, with genotyping quality
(Q) >20 and significative effect predictors, SIFT and PolyPhen. We analysed genetic
variants with an important functional consequences which were present in controls or
cases. Specifically, we focused in the analysis of splicing variants, missense variants,
variants in mature microRNAs coding sequences and stop variants. Finally, we verified
the stop variants by Sanger sequencing.
Results: Exome sequencing approximately generated 1,000 GB data which after
the bioinformatics analysis became around 2,000 GB in total. It was identified 21,822
SNPs in controls and 17,238 in cases with a functional effect, present only in controls or
cases that meet quality criteria. In particular, 160 and 132 SNPs were splicing variants
which were identified in controls and cases, respectively. While 1,817 and 1,614 SNPs
were missense variants in controls and cases, respectively. Furthermore, 23 SNPs were
identified as mature microRNA variants in controls and 11 SNPs in cases. Finally, we
identified 102 SNPs as stop variants in controls and 50 SNPs in cases.
Conclusions: We have identified a large number of genetic variants, including lowfrequency
and rare variants, which may be involved in the development of T2DM or in the
protection from it. In order to stablish the true genetic variants involved in the disease we
will need to validate them by different strategies, replication in a large sample of controls
and diabetics as well as carrying out functional studies.